The nonLTR retrotransposon LINE-1 (L1Hs) is actively transposed in the human genome where its insertion has been associated with mutations causing a variety of genetic diseases and several tumors. About 100,000 copies of L1Hs occur in primate genomes but most of these are nonfunctional. Current estimates suggest that there may be on the order of 50 active elements in the human genome. Our work is directed toward understanding the mechanism and regulation of L1Hs transposition. Recent experiments have extended our efforts on the biochemistry of the proteins encoded by the two L1Hs open reading frames: ORF1 and ORF2. The 1275 amino acid long protein encoded by ORF2 is synthesized in yeast cells provided with an appropriate vector and is an active reverse transcriptase. Interestingly, the L1Hs reverse transcriptase synthesized in yeast is dependent on a primer (oligo dT) in copying poly rA template but is primer independent when the template provided is poly rC. Using deletion constructs, we have demonstrated that approximately 480 amino acids (residues 470 to 951) are sufficient for enzymatic activity. Deletions altering one or more amino acids in the so-called Z region (470-476) indicate that this segment is required for activity. Z is highly conserved among Line elements in many species although it is not one of the regions identified as being conserved in all reverse transcriptases. The protein, p40, encoded by ORF1, occurs in large ribonucleoprotein complexes in association with L1Hs RNA in teratocarcinoma cells (see last year's report). These complexes dissociate upon treatment with ribonuclease or high salt; the RNA is lost but smaller, multimeric complexes of p40 accumulate. The p40 multimers isolated from teratocarcinoma cell extracts with high salt bind L1Hs RNA in vitro. Using an assay that depends on the RNA with RNaseT1 and immunoprecipitation, two specific p40-binding oligoribonucleotides were identified. Our experiments demonstrate that p40 is a unique sequence- specific RNA-binding protein with specificity for single-stranded RNA.